Energetic determinants of tyrosine phosphorylation of focal adhesion proteins during hypoxia/reoxygenation of kidney proximal tubules

Anaerobic mitochondrial metabolism of α-ketoglutarate and aspartate or α-ketoglutarate and malate can prevent and reverse severe mitochondrial dysfunction during reoxygenation after 60 minutes of hypoxia in kidney proximal tubules. The present studies demonstrate that, during hypoxia, paxillin, focal adhesion kinase, and p130^cas migrated faster by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their phosphotyrosine (pY) content decreased to ∼5% of that in oxygenated tubules without changes in total protein, and the normally basal immunostaining of β1 and α6 integrin subunits, pY, and paxillin was lost or markedly decreased. During reoxygenation without supplemental substrates, recovery of pY and basal localization of the focal adhesion proteins was poor. α-Ketoglutarate and aspartate, which maintained slightly higher levels of ATP during hypoxia, also maintained 2.5-fold higher levels of pY during this period, and promoted full recovery of pY content and basal localization of focal adhesion proteins during subsequent reoxygenation. Similarly complete recovery was made possible by provision of α-ketoglutarate and aspartate or α-ketoglutarate and malate only during reoxygenation. These data emphasize the importance of very low energy thresholds for maintaining the integrity of key structural and biochemical components required for cellular survival and reaffirm the value of approaches aimed at conserving or generating energy in cells injured by hypoxia or ischemia.